Accurate chromosome segregation is essential for the propagation of species and the viability of cells, and is driven by a complex microtubule-based structure called the spindle. Intensive biochemical, genetic, and proteomic efforts provide an extensive catalogue of proteins that participate in spindle organization and spindle-dependent chromosome movement. However, these efforts don't reveal the molecular mechanisms that ensure faithful chromosome segregation in mammalian cells. Recently, we showed that the most common cause of chromosome mis-segregation in human tumor cells is the persistence of kinetochore- microtubule (k-MT) attachment errors. This indicates that the most important mechanism ensuring faithful chromosome segregation is the fine-tuning of k-MT attachment stability, yet our understanding for how k-MT attachment stability is regulated during mitosis is starkly incomplete. We don't understand how the stabilizing and destabilizing components work in concert to generate a coherent k-MT attachment stability at different phases of mitosis. We also don't understand how aneuploidy influences genome stability and cell survival. Based on models generated during the current funding period, it is our goal in the forthcoming funding period to combine biochemical methods and live cell imaging to test models and determine the mechanisms of the regulation of K-MT attachments in mitosis. Understanding these mechanisms could lead to new therapeutic approaches for cancer treatment. RELEVANCE (See instructions): Errors in chromosome segregation cause aneuploidy that causes birth defects and is commonly associated with advanced stage cancer. The goal of the experiments proposed here is to identify the proteins and determine the mechanisms responsible for high fidelity chromosome segregation in human cells. Data generated from this work will provide insight into mechanisms of aneuploidy in tumor cells and may reveal strategies for therapy of chromosomally unstable aneuploid tumors.